Bovine spongiform encephalopathy (BSE or mad cow disease) of cattle and scrapie of sheep are fatal, non-inflammatory neurodegenerative diseases caused by prions and are characterized by a long incubation period. In humans, Creutzfeldt-Jakob disease (CJD), Gerstmann-Straussler-Scheinker syndrome (GSS), fatal familial insomnia and kuru belong to this category of TSEs.
Although scrapie, the prototype of the family of TSEs, in sheep and goats has been known for over 200 years (Pattison, 1988) and has been diagnosed world-wide (with the exception of New Zealand and Australia), it is only since 1986 that BSE has been described in cattle in the UK. By Jan. 1998, there had been 170,259 confirmed cases of BSE in Great Britain and there may exist a great number of cases of not yet overt (“silent”) BSE. BSE probably emerged because scrapie-contaminated sheep offal had been included in cattle feeding-stuff via meat and bone meal and newly infected cattle material was then recycled (Wilesmith et al., 1991). This mechanism is quite plausible since ovine scrapie could be transmitted experimentally to several animal species, including cattle (Hourrigan, 1990; Gibbs, 1990).
Alternatively, recycling of offal from a rare case of spontaneous BSE for cattle feedstuff could also have led to the BSE epidemic. Moreover, the number of cattle in the UK with BSE reported annually is declining after the ban on feeding meat and bone meal in 1988.
Brain homogenates from cows with BSE produce, after inoculation of mice, a characteristic pattern of brain lesions in mice. Also, characteristic incubation periods in inbred lines of mice are seen. This is identical to the pattern elicited by brain tissue from individuals who recently have died from new-variant Creutzfeldt-Jakob disease (nvCJD; Bruce, 1997). The conclusion is that the BSE agent is identical to the nvCJD agent. Through 1996, this variant has caused the death of 35 young Britons and one Frenchman (Will et al., 1996).
There is also concern that the BSE strain that seems to be transmissible to humans may have infected sheep, where it could produce a disease hardly distinguishable from scrapie. When its ominous strain-specific properties are maintained across the species barrier, sheep BSE may be a threat to human health, although scrapie by itself does not seem to transmit to humans. Indeed, BSE agent has been transmitted experimentally to sheep by the oral route (Foster et al., 1993) and thus could have the potential to infect sheep under field conditions. With the exception of a bioassay in mice, no diagnostic method is available to discriminate between BSE and scrapie in sheep at present.
Thus far, the only known component of the infectious prion is an abnormal, disease-causing isoform of the “normal” prion protein (PrP) called PrPSc or aberrant prion protein. PrP, or normal prion protein, is ubiquitous in mammalian cells in a benign, cellular conformation (PrPC) and is encoded within a single exon as a protein of about 250 amino acid residues (FIG. 1) (SEQ ID NOS:1-6). The PrP gene has been cloned and sequenced from a variety of species, and there is a high degree of structural and organizational homology between mammalian PrP sequences (Schatzl et al., 1995). PrPs in many mammals have a 22-24-residue long N-terminal signal sequence as well as a 22-24-residue long C-terminal signal sequence for attachment of a GPI-anchor. This glycosylphosphatidylinositol linkage is a fairly common means of anchoring proteins to membranes of eukaryotic cells. Further structural characteristics of the mature protein (of 206-210 amino acid residues) are one disulfide bond and two sites for Asn-linked glycosylation.
PrPSc originates from the normal cellular isoform (PrPC) by a post-translational process since the amino acid sequence of PrPSc is identical to that predicted from cDNA or genomic nucleic acid sequences. Glycosylation patterns are also identical between PrPC and PrPSc. Moreover, Caughey and Raymond (1991) demonstrated that PrPSc is made from a cell surface precursor that is identical to the normal PrP. PrPSc differs from the normal, membrane-bound cellular prion protein by its relative protease resistance. Treatment with proteinase K (PK), for instance, results in complete proteolysis of PrPC, whereas in PrPSc, the N-terminal part is removed before the amino acid at position 90 (human numeration) (SEQ ID NO:1). The protease-resistant core left is designated PrP27-30 after its electrophoretic behavior in SDS-PAGE as a protein molecule with Mr=27-30 kDa, and this molecular species retains full infectivity.
Further distinguishing features of PrPSc are its thermal stability, a strong tendency to aggregate and insolubility in non-denaturing detergents, apparently connected with a different molecular structure. All attempts to identify a post-translational chemical modification that features in the conversion of PrPC into PrPSc have been unsuccessful.
The lack of a molecular explanation for the observed differences between PrPSc and PrPC led to the proposal that they must differ in conformation. Indeed, Fourier transform infrared spectroscopy detected a content of 43% of β-sheet and 30% of α-helix structure for purified hamster PrPSc and an even higher P-sheet content of 54% for PrP27-30. On the other hand, a low content of β-sheet structure and a high α-helix content of 42% was found in PrPC, suggesting differences in secondary structure between the aberrant and normal forms of PrP (Pan et al., 1993).
Due to its better solubility and the availability of recombinant forms of PrPC, the three-dimensional structure of mouse PrP (121-231), involving three α-helices and a short antiparallel P-sheet, could be established by NMR (Riek et al. 1996). In the mature murine PrPC (23-231), this segment seems to have the same fold (Riek et al., 1997). Also, the spatial structure of recombinant hamster PrP (29-231) has been examined (Donne et al., 1997).
A species barrier for prion infection has been convincingly documented and found to vary widely depending on the pair of species involved and the direction of transmission. A structural basis for this species barrier is theoretically related to part or all of the amino acid replacements between the PrP of a given pair of species (Billeter et al., 1997).
Within species, genetic polymorphism in the PrP gene has been found, for example, with mice, humans and sheep. In sheep, amino acid substitutions in PrP at a few different positions were found to correlate with different predispositions for the development of scrapie (Laplanche et al., 1993; Hunter et al., 1994; Belt et al., 1995; Bossers et al., 1996).
Studies of scrapie in goats and mice demonstrated reproducible variations in disease phenotype (length of incubation times and pattern of vacuolation) with the passage of prions in genetically inbred hosts (Bruce and Fraser, 1991). The distinct varieties or isolates of prions were called “strains.” Safar et al. (1998) made plausible that the biological properties of prion strains are enciphered in the conformation of PrPSc and that strains represent different conformations of PrPSc molecules. Infection of Syrian hamsters with eight different hamster-adapted scrapie isolates produced PrPSc molecular species which, isolated from brains in the terminal stages of disease, differed with respect to protease resistance and unfolding behavior under denaturing conditions. Differences in glycosylation have also been proposed as “strain-specific” properties (Collinge et al., 1996).
Animals and humans lack a TSE disease-specific immune response and TSE diagnosis is based mainly on histopathological examination, which relies on the observation of neuronal degeneration, grey matter vacuolation (the spongiform change) and astrocytosis. A distinguishing feature of TSEs is the accumulation of aberrant protein (PrPSc) in the brain under continuing biosynthesis of the normal cellular PrPC. Species differences exist, however, since the relative accumulation of PrPSc in brains of hamster and mouse is approximately ten times as high as in the ruminant. Unlike the normal PrPC, PrPSc can aggregate into amyloid-like fibrils and plaques and is a major component of brain fractions enriched for scrapie activity. Therefore, a more specific diagnosis of TSEs is detection of PrPSc, either in situ, e.g., by immunohistochemistry, or in tissue homogenates, e.g., by Western blot.
Several poly- or monoclonal antibodies to PrP have been described. The antisera were raised in mice, hamsters, rabbits and PrP null mice and as immunogens, peptides (as linear epitopes), purified and formic acid-treated PrPSc from mice, hamster or sheep and recombinant PrP are being used. However, except for one case (Korth et al., 1997), there have been no antibodies developed that can discriminate between native forms of PrPC and PrPSc, and such antibodies cannot likely discern the difference between prion strains.
By Western blotting or immunohistochemistry, PrPSc could be detected in sheep in brain, spleen, tonsil or lymph node material and even in a preclinical stage of scrapie (Schreuder et al., 1998). However, in BSE-infected cattle, PrPSc could not be detected outside the central nervous system, not even when clinical symptoms were present.
The intriguing mechanism of prion replication is not fully understood. According to the prevailing theory, the infectious PrPSc acts as a template in the replication of nascent PrPSc molecules. In other words, PrPSc imposes its own conformation upon the cellular form PrPC or an intermediate form. A thus far unknown protein X may function as a molecular chaperone in this formation of PrPSc (Prusiner et al., 1998).
Because of the connection between BSE and the nvCJD, and the possible transfer of BSE to other species including sheep, there is a need to monitor slaughter cattle and sheep for the presence of aberrant prion protein before the meat and meat products enter the human and animal food chain or into pharmaceuticals prepared for human and animal use. Mass screening of sheep and cattle should also be of help in view of eradication programs of scrapie and BSE. Moreover, human blood and blood products may form a health threat on account of possible contamination with blood of CJD patients and the recent occurrence of the nvCJD. For these monitoring purposes, a detection method for aberrant prion protein has to be developed that should be both fast, sensitive, reliable and simple.
Bioassays for PrPSc in which different doses of the analyte are administered to target animals are generally regarded a gold standard but otherwise are cumbersome and costly. Moreover, their quantitative character is limited by a high variation. Immunohistochemical (IHC) approaches are very useful insofar as the presence of the analyte is directly made visible in the infected tissue. In particular, testing the sample by histology or cytology allows a morphological comparison of healthy and diseased cells or tissue. Also, the presence of PrPSc can be indicated in a preclinical phase. However, and in general, histological or cytological methods are not quantitative and hardly applicable on a large scale.
For the diagnosis of TSEs founded on the demonstration of PrPSc in infected tissues and for the assessment of PrPSc itself, several methods have been described and all are on an immunochemical basis. Most of these tests have been developed and used for research-like purposes, for instance, in order to quantify PrPSc during purification procedures. In some cases, calibration was with recombinant PrP (hamster or mice) or with PrPSc, purified from scrapie-infected brains. Otherwise, responses were expressed as a function of mg tissue equivalents; in this way, sensitivity could also be assessed by the minimum amount of tissue required for the PrPSc detection.
ELISA systems were designed for detection of PrPSc, isolated from brains of scrapie-affected mice and hamsters (Kascsak et al., 1987) and PrPSc from murine brain and spleen (Grathwohl et al., 1997). In these assays, the PrPC fraction was beforehand removed by PK treatment and the purified and solubilized analyte was directly coated onto the microtiter plate. Solubilization of PrPSc was by treatment with SDS or extraction with 77% formic acid, drying and resuspension in buffer (Kascsak et al. 1987). The denaturing action of formic acid was found to enhance the antibody response to PrPSc considerably, compared to untreated or SDS-treated material. In this ELISA, rabbit antiserum to the mouse scrapie strain ME7 PrPSc was used.
Also, successive solubilization of purified PrPSc by boiling in SDS, precipitation in cold methanol and sonication in 3-4 M guanidine thiocyanate (gdnSCN) (Grathwohl et al., 1997) apparently enhanced coating efficiency and/or epitope density under the denaturing action of gdnSCN. On the other hand, dissolving PrPC in SDS appeared to inhibit adsorption of PrPSc onto the polystyrene microtiter plate. Although Grathwohl et al. (1997) state that their method could be a basis for a sensitive screening method for PrPSc in crude tissue extracts, their extraction and purification steps are impracticable and time consuming (over 22 hours). The sensitivity for brain tissue was such that PrPSc could be detected in 39 mg brain equivalents; the corresponding figure for spleen tissue amounted to 313 mg. Bell et al. (1997) report comparative research of five research centers of in-house immunohistochemical methods for the detection of aberrant protein in CJD by histological staining of brain tissue sections. As to the use of gdnSCN, two of the five centers employ, in addition to formic acid, gdnSCN to pretreat their tissue sections to inactivate the prion agent to allow further processing of the tissues without the danger of infection. However, all over, the value of the addition of gdnSCN is questioned and, in the opinion of one center, it even increases background in histology. Effective decontamination of prion-containing CJD material is also shown in WO 98/32334.
A sandwich type of ELISA was used to monitor the bioproduction of recombinant hamster PrP_(90-231), the protease-resistant core of PrPSc (Mehlhom et al., 1996). As a capture antibody, the Fab fragment of mAb 3F4 was coated onto the microtiter plate. This antibody was raised against hamster scrapie strain 263K and reacts with hamster, human and feline PrP. As the second antibody, mAb 13A5 (to scrapie hamster PrPSc) was used. Samples from the different stages of purification were measured in this ELISA. However, the practical conditions under which PrPSc, in order to be detected as an antigen, is brought into an unfolded state by chaotropic agents like 3-4 M gdnSCN, are not compatible with the immunochemistry of a sandwich type of ELISA.
Prusiner et al. (1990) used an enzyme-linked immunofiltration assay (ELIFA) that combines the properties of an immuno-dot blot and ELISA technique. By this method, both PrPC and PrPSc in scrapie brain homogenates of hamsters could be quantified against a standard curve of known amounts of purified hamster PrP27-30 (0.06-4 ng). Brain homogenates, diluted in buffer with 1 M gdnSCN and 0.05% Tween 20, were applied in 5 μl quantities to nitrocellulose membrane in a manifold filtration unit. Sequential steps for immunocomplex formation with mAb 13A5 and conjugation of enzyme were also done on this membrane. For detection, dots were cut out with a puncher and placed into a microtiter plate in which color was developed. Under these conditions, immunoreactivity of the dissociated and (partly) unfolded PrPSc is indistinguishable from that of PrPC and in this way total PrP was measured. For the determination of the PrPSc fraction, the homogenate was treated with PK prior to the ELISA and PrPC content was calculated by subtracting the PrPSc from the total PrP.
Oesch et al. (1994) refined this ELIFA method. Samples were applied on nitrocellulose filters in the ELIFA apparatus, procedures hereafter among which a two-hour-preincubation in 4 M gdnSCN to render the aberrant protein sensitive to protease digestion, and substrate binding to mAb 13A5, up to and including binding with the enzyme, were done on the membrane taken out of the apparatus. For detection, membranes were placed back in the ELIFA apparatus and reacted with substrate solution. Finally, the reaction mixture was pulled through into an ELISA plate placed underneath and color development was measured. This whole procedure took over 20 hours.
Immuno-dot blotting was used by Serban et al. (1990) for the post-mortem diagnosis of Creutzfeldt-Jakob disease in humans, scrapie in sheep and scrapie-infected hamsters and mice. Direct spotting of a rather impure analyte on, e.g., nitrocellulose filters instead of adsorption of a purified fraction of it onto the plastic surface of microtiter wells produces a more robust ELISA variant. This qualitative test was based on the intensified immunoreactivity of PrPSc-containing amyloid plaques after treatment with 3 M gdnSCN and the protease resistance of the PrPSc isoform.
Brains were extracted in detergent-containing lysis buffer and 4 μl amounts were spotted onto nitrocellulose membranes. Immunoreactivity of the spotted material after successive treatment with PK and 3 M gdnSCN was conclusive for the presence of PrPSc and confirmation of CJD and scrapie. Rabbit Ab R075 (to purified hamster PrP27-30) was able to detect PrP in the above four species. Out of a total of 28 human brain samples, nine cases found positive by this method were also either defined as CJD or GSS by both clinical diagnosis and a histopathological examination. For two cases found positive by the blot procedure, histopathologic results were not available. The negative results of histopathology for CJD or GSS on the remaining 17 cases coincided also with no indication for PrPSc with the immuno-dot blot assay. In 12 histologically confirmed cases of natural scrapie in sheep, PrPSc was detected with the immunoblotting technique in the brains of 11 sheep. There are variations in the distribution of PrPSC in the brain of scrapie-affected sheep, since PrPSc was found in the spinal cord, cerebellum and pons/medulla of two sheep, but one sheep also had PrPSc in the frontal and occipital cortex and the thalamus. This means that sampling of brain tissue could lead to false negatives due to regional variations in PrPSc content. The detection limit of this method for brain extracts of scrapie-infected hamsters and mice ranged from 5-132 mg tissue equivalents, because these amounts still gave clearly visible spots. The duration of the test was, apart from an overnight incubation step, six hours.
Safar et al. (1998) developed a conformation-dependant fluorescent-ELISA that can discern various prion strains of hamsters. The assay detects a region of PrPSc that, while exposed in normal PrPC, becomes folded in the PrPSc molecule. Eu-labeled mAb 3F4 that reacts with a region of PrPSc only after unfolding in 4 M gdnHCl and heating at 80° C. for five minutes, was used in this assay. The immunoreactivity of the antibody to the denaturated region, as reflected by the fluorescence signal, is much higher than it is to PrPSc in its native form. The authors developed an algorithm that takes into account that the immunoreactivity of antibody to denatured PrP in a sample of an affected brain is the summation of enhanced immunoreactivities of PrPSc and PrPC during the transition from the native to the denatured states. Knowledge of the enhancement of immunoreactivity for PrPC during denaturation was a prerequisite for this approach. For this purpose, calibration curves with different concentrations of purified PrPC were constructed. It appeared that also PrPC showed an enhanced immunoreactivity in 4 M gdnHCl, compared to its native state, albeit in a moderate way (≦1.8×). From the algorithm and the measurements of a native as well as a denatured sample, the content of PrPSc could be calculated. Although this method was validated for the determination of hamster brain, the authors aim at using it also for the detection of other mammalian prions, including human. In order to improve the detection threshold of the assay, they introduced an initial step to selectively precipitate PrPSc from raw material with sodium phosphotungstate. In combination with this sample pretreatment, the final sensitivity of the assay could be made high. The sensitivity limit is less than or equal to 1 ng/ml (100 pg) of PrPSc. The test, however, is still far from lending itself to large-scale use in view of too much labor and long incubation times.
Capillary electrophoresis was adapted by Schmerr et al. (1995, 1996, 1998a), as a diagnostic, immunochemical assay for scrapie. The authors claim a high sensitivity (approximately 135 pg PrPSc) of their test by measuring laser-induced fluorescence of a PrP-derived fluorescein-labeled peptide after its separation by free-zone capillary electrophoresis. In a preceding competition step, this peptide was displaced from a preformed complex of the peptide and an antibody directed to the unlabeled peptide in competition with the analyte (PrPSc). Beforehand, PrPC had been removed from the analyte solution by PK treatment. The concentration of rabbit antiserum for complex-preformation was chosen so that the antibody would be limiting in the assay (adjustment to 50% of the maximum amount of immunocomplex). Four anti-(prion)-peptide antisera were prepared and evaluated. Assays using antisera to the peptides spanning mouse amino acid position 142-154 (SEQ ID NO:4) and 155-178 (SEQ ID NO:4) differentiated scrapie-positive sheep from normal animals. In spite of the high sensitivity of this method, sample processing is time consuming (approximately 24 hours) and cumbersome since PrPSc from brain stem has to be concentrated and purified through steps like ultracentrifugation and HPLC.
Western blotting (WB), in combination with SDS-PAGE, is also a suitable technique for diagnosis of TSEs and a variety of different extraction procedures and Western blotting methods has been described (Race et al., 1992; Beekes et al., 1995).
Usually, PrPC is extracted from tissues with detergents that solubilize this membrane-bound protein in a mixed micelle. However, PrPSc in the presence of detergents, aggregates and, therefore, is not solubilized but can be spun down by ultracentrifugation. PrPSc aggregates dissociate in monomers under the denaturing conditions of heating in SDS solution with β-mercaptoethanol. In this way, PrPSc is electrophoretically (SDS-PAGE) indistinguishable from PrPC, unless a preceding treatment with PK has been applied. This proteolytic treatment removes PrPC and leaves PrP27-30, the truncated form of PrPSc.
Race et al. (1992) could find PrPSc in every brain of eight sheep that were histologically positive for scrapie and even in brains of clinically positive sheep that were not diagnosed as scrapie-positive by histology. For detection, antipeptide antibodies to residues 89-103 (SEQ ID NO:4) and 218-232 (SEQ ID NO:4) of the mouse PrP sequence were used. Apparently, the amount of tissue required to visualize PrPSc varied among sheep from <2 to 200 mg equivalents of brain tissue. Also, PrPSc was found in spleens and lymph nodes in seven of eight sheep that had the protease-resistant form detected in brain homogenates.
One method based on WB was officially approved by the European Union (EU) and the World Organization for Animal Health (OIE) for BSE and scrapie diagnosis (Bradley et al., 1994). A minimum amount of 2 mg equivalent of infected scrapie brain allows detection of the PrP27-30.
Above-identified assays have never been used in large screening efforts for the detection of aberrant prion protein, neither in animals nor in humans.
Thus far, two commercial assays have been announced. In 1997, the Swiss company Prionics Inc. launched its “BSE Western Test” intended for mass screening of slaughter cattle. A modified and optimized Western blot method was used to detect the proteinase K-resistant PrP27-30 in bovine brain stem. For immunodetection, mAb 6H4 was used, developed by immunizing PrP-null mice with recombinant bovine PrP. This antibody recognizes residues 147-155 (SEQ ID NO:5) of the bovine sequence as a linear epitope in native PrPC and denatured PrPSc; this sequence is also recognized in sheep, human, pig and mouse. Incubation with anti-mouse IgG coupled to alkaline phosphatase and detection of the enzymatic product by chemiluminescence were the final steps of the assay. This test requires an incubation step with PK and detects PrP27-30. Reliability is strengthened by the Western blot documentation of the decrease in size (internal control) of the prion protein from 30-33 to 27-30 kDa. The test can be done within hours, and the expectation is that subclinical BSE in post-mortem brains may be detected.
Also, in 1997 the Irish Company Enfer Scientific Ltd. announced the development of a BSE post-mortem test. This immunoassay intended for mass screening uses a PrP antipeptide antiserum to detect PrP27-30 in samples of brain tissue of cattle after removal of PrPC by PK treatment. Immunodetection was enhanced by chemiluminescence. Their claims are a result within four hours after receipt of samples and a capacity of 14,000 cattle a day and, moreover, the catching of asymptomatic animals.
However, these two commercial tests, although claiming high sensitivity in detecting the aberrant protein, and thus claiming to have a low number of false-negative results, suffer from the low specificity associated with the claimed high sensitivity. When using the above tests, one, therefore, runs an increased risk of falsely identifying a negative sample as false positive, thereby falsely identifying an animal as positive. For example, Switzerland slaughtered herds in which one or more cases of BSE had been confirmed. The “Swiss reference laboratory for animal TSE” examined the brains of these 1761 apparently healthy cattle by an immunohistochemical method for signs of BSE and six positive cases were detected. Also. Prionics Inc. tested these 1761 cattle brains by their “BSE Western Test.” Four positive outcomes were identical to the ones found by the reference laboratory, the other two were indicated as negative and, moreover, two other cattle were found positive by Western blotting. Thus, a total of eight positive reactors were found, four of which overlapped. These eight were re-examined in the laboratory of Dr Kretzschmar (University of Gottingen) and in addition to the four undisputed cases, one of the two questionable cases identified by the reference laboratory could be confirmed (info: New Scientist, 1998, July 4 and Internet). Prionics, for example, scored 0.1% false positives, indicating that in one of every thousand cases, a sample causes a false alarm due to false positivity.
Tests scoring false-positive results (being, in general, not specific enough) have other consequences than tests scoring false-negative results (being, in general, not sensitive enough).
“False negative” means that, in essence, a positive sample from a positive individual is scored negative and, thus, is not suspected of having a TSE, while in truth, the individual does have a TSE. A false-negative diagnosis thus results in missing positive cases.
For humans, “false negative” means that no diagnosis of TSE is made where the human actually has a TSE. This causes a wrong prognosis being established and wrong treatment being given, until a second test is done.
For animals, especially in those cases where slaughtered animals are tested, “false negative” means that no diagnosis of TSE is made where the animal was actually infected and possibly capable of spreading the disease without having been noticed. Meat and other products from such a false-negative animal may contain aberrant prion protein. Such meat and meat products will be traded and eaten and can thus be a source for further infection, notably of humans who even falsely trust that the animal has been tested well and the meat or meat product bears no risk.
“False positive” means that, in essence, a negative sample from a negative individual is scored positive, and thus is at least suspected of having a TSE, while in truth, the individual does not have a TSE at all, but possibly another condition.
For humans, “false positive” means that a false diagnosis of TSE is made, here again resulting in false prognosis and in faulty treatment. If the individual is not treated well as a consequence of the misdiagnosis, his or her possible other disease condition (the symptoms of which, for example, gave rise to the decision to test for TSE) receives no proper treatment.
For animals, “false positive” means that a false diagnosis of TSE is made, however, since TSEs are notifiable diseases that in general are met with strict eradication measures, the animal shall, at least in most Western countries, be killed and destroyed. Furthermore, the herd from which the animal originated runs the same risk of being destroyed when the diagnosis is not corrected. For the slaughterhouse, it might mean that special laborious decontamination actions have to be implemented, which mean temporary interference of use of the facilities and thus considerable loss of productivity. Additionally, the country where the animal or herd is falsely diagnosed for having a case of TSE among its animals will be met with export restrictions. It goes without saying that, especially when the country has no (present) reported cases of TSE, such a false-positive diagnosis is highly detrimental for the country's position on foreign markets for animal products.
Understanding the above risks associated with false-negative or false-positive diagnoses becomes even more complicated when one understands that, in general, the level of false positives scored by a diagnostic method or test is inversely related to the number of false negatives scored by the same test. It is an old diagnostic truth that, in many instances, a very sensitive test (having low numbers of false negatives) cannot be very specific (and, thus, has a relative high number of false positives) and vice versa. However, and especially for mass screening tests that do not comprise histology or cytology, and wherein many samples need to be tested, tests having both high sensitivity and specificity are desired.